Deciphering the spectrum of cutaneous lymphomas expressing TFH markers

T-follicular helper (TFH) markers are expressed in the microenvironnement of marginal zone B-cell lymphoma (MZL), and in lymphomas arising from TFH-cells, sometimes making the differential diagnosis difficult. In the skin, the “TFH-spectrum” is poorly defined, going from primary cutaneous lymphoproliferative disorder with small/medium CD4+ T-cells (SMLPD) to cutaneous localizations of systemic angioimmunoblastic T-cell lymphoma (cAITL), and may pass through intermediate forms (primary cutaneous T-follicular helper derived lymphoma, not otherwise specified (PCTFHL,NOS)). We retrospectively analyzed 20 MZL, 13 SMLPD, 5 PCTFHL, and 11 cAITL clinically, histologically, and molecularly, to define tools to differentiate them. Characteristics that might favor the diagnosis of MZL over SMLPD are: multiple skin nodules (p < 0.001), nodular architecture (p < 0.01), residual germinal centers with follicular dendritic cell network (p < 0.001), monotypic plasma cells (p < 0.001), and few staining with PD1 (p = 0.016) or CXCL13 (p = 0.03). PCTFHL and cAITL presented as multiple (p < 0.01) lesions, in older patients (p < 0.01), with systemic symptoms and/or biological alterations (p < 0.01). Immunophenotypic loss of T-cell markers (p < 0.001), BCL6 (p = 0.023) and/or CD10 staining (p = 0.08), and a higher proliferative index (≥ 30%, p = 0.039) favoured these diagnoses over SMLPD. Pathogenic variants were observed by genomic sequencing in 47% of MZL (TNFAIP3 (32%), EP300 (21%), NOTCH2 (16%), KMT2D (16%), CARD11 (10.5%)), 8% of SMLPD (TET2), 40% of PCTFHL (SOCS1 (20%), ARID1A (20%)) and 64% of cAITL (TET2 (63.6%), RHOA (36.4%), NOTCH1 (9%)). This study characterizes the various clinical and histological features between cutaneous lymphomas expressing TFH markers and highlights the value of the interest of screening for genomic mutations in difficult cases.

entities which present a distinct aggressiveness and evolution. Furthermore, molecular alterations of PCTFHL are unknown.
TFH-cells are also find in the microenvironnement of B-cell lymphomas, especially marginal zone lymphomas (MZL) 6,[8][9][10] . The clinicopathological characteristics of cutaneous MZL are close to some cutaneous T-cell lymphomas with a TFH phenotype, in particular primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder (SMLPD) 10 . The distinction between MZL and SMLPD is sometimes difficult and clonality study is not always discriminatory due to cases without monoclonal rearrangement or with both B (BCR) and T-cell receptor (TCR) rearrangements 7 .
We retrospectively described a series of 49 cutaneous lymphomas with TFH-markers expression, including cases with TFH expression in tumoral cells and cases with TFH hyperplasia in the microenvironnement, to characterize them at a clinicopathological and molecular level and highlight tools to differentiate them.

Patient selection. TFH-cells arising lymphomas (SMLPD and cutaneous localizations of systemic AITL)
and cutaneous MZL diagnosed from March 2017 to March 2019 in agreement to the 2016 WHO classification of hematologic malignancies were retrieved from the pathological department of the Lyon Sud University Hospital, France. Intermediate forms between SMLPD and AITL, so-called PCTFHL, were also retrieved.
Inclusion criteria for MZL, PCTFHL and SMLPD were the presence of a monoclonal B-or T-cell population, to limit the inclusion of reactive lymphoid hyperplasia, and enough FFPE material available. All cases had to have benefited from an extensive assessment including either a CT-scan or chest X-ray and abdominal and pelvic ultrasound, to exclude systemic lymphoma with secondary involvement of the skin. MF and/or Sezary syndrome were ruled out by clinicopathological correlation and expert opinion from the French cutaneous lymphomas study group (GFELC). Concerning cutaneous localizations of AITL, cases were included only if they had a known history of systemic AITL, proved by a lymph node biopsy.
Clinical, biological, and radiological data. Clinical data were retrospectively collected. It included: age at diagnosis, sex, medical history, clinical presentation (number of lesions, localization, systemic symptoms, and lymphadenopathy), biological data (blood count, borreliosis serology), imaging data, received treatments and evolution. Patients were divided into two groups: (i) "indolent" (spontaneous disappearance and/or no relapse after local treatment); or (ii) "refractory/relapsing" (relapse/persistence after initial local treatment requiring chemotherapy or radiotherapy).
Clonality study and targeted next generation sequencing (TNGS). Clonality study was performed according to the EuroClonality/BIOMED-2 protocol 22 . IGH and IGK assays were used for BCR study, with the following primers: FR1, FR2, FR3, DH-JH, Ig kappa, and Ig lambda. TCR Clonality was assessed using the BIOMED-2 primer sets for TCR gamma (TCRG). TNGS have been performed as previously reported 11,23 . The applied panel of 47 genes is detailed in Suppl. Table S2. Only pathogenic variants with a minimal depth of 700× were retained. Supplemental quality data are available in Suppl. Table S4.

Results
Sixty-seven cases corresponding to 27 MZL, 21 SMLPD, 8 PCTFHL and 11 cAITL were retrieved. Seven MZL were excluded due to the presence of a systemic MZL. Eight SMLPD were excluded: 3 because the material was exhausted, 4 due to the absence of any monoclonal T-cell population, and one because further evaluation led to reclassification as MF. Three PCTFHL were excluded, two due to the absence of TCR clonal rearrangement, and one due to the presence of Sezary cells in the blood, leading to reclassification as Sezary syndrome. Twenty MZL, 13 SMLPD, 5 PCTFHL, and 11 cAITL were finally included.
Among PCTFHL, the median follow-up was of 20 months. The case no 5 with a monoclonal circulating T-cell population responded completely under topical corticosteroids and was considered indolent. Three were refractory/relapsing patients; one received topical corticosteroids with a remitting relapsing course (median www.nature.com/scientificreports/ follow-up of 50 months), one received radio-chemotherapy (gemcitabine), and the last received methotrexate. The fifth was lost to follow-up. All cAITL were treated by chemotherapy (9 CHOP: cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) and 2 GEMOx: gemcitabine, oxaliplatin).
Clonality study (Tables 2, 3, 4). With the presence of a monoclonal population being inclusion criteria, BCR rearrangements were present in all MZL and TCR rearrangements in all SMLPD and PCTFHL. The same clone was found in the skin biopsy for the PCTFHL case with a circulating T monoclonal population.
Concerning cAITL, 5 presented a monoclonal T-cell population, 2 presented both BCR and TCR rearrangement, results were undetermined in 3 cases, and one did not display any monoclonal population (AITL no 9).
Two SMLPD and one MZL presented both BCR and TCR rearrangements. The 2 SMLPD presented characteristics typical of SMLPD: unique nodules, 66-80% of T-cells, absence of FDC network, 5-20% polytypic plasma cells, medium PD1/CXCL13 staining (10-30%). The MZL case presented characteristics typical of MZL: multiple lesions, majority of B-cells (60%), nodular pattern, residual germinal centers, 25% of plasma cells with lambda monotypia, low PD1/CXCL13 staining (10%). Tables 3, 4). The TNGS analysis (Table 3) was performed in all cases except for 1 MZL and 1 SMLPD due to a poor DNA quality. Pathogenic variants were found in 9 MZL (47%), 1 SMLPD (8%), 2 PCTFHL (40%) and 7 cAITL (64%). Pathogenic variants found in MZL were: TNFAIP3 (  Plasma cells were few (< 10%) but presented a kappa monotypia. The diagnosis of MZL was confirmed thanks to molecular data; the presence of a monoclonal B-cell population, and pathogenic variants of ITPKB, NOTCH2, TNFAIP3, and KMT2D genes using TNGS. Molecular analysis had also been performed in addition to the skin tissue in lymph node biopsies of 4 AITL (Table 4). Cases no 3, 7 and 10 presented a similar monoclonal T population both samples, case no 9 had had a TCR gamma chain rearrangement with an undetermined ratio in the skin biopsy, and a true T monoclonal , which were polytypic for kappa and lambda, and located outside of the proliferation in this particular case, but scattered among T-cells in most SMLPD cases; (H) (PD1, ×20), (I) (CXCL13, ×20), (J) (BCL6, ×20) and (K) (CD10, ×20): huge expression of TFH markers except for CD10 which was negative (PD1 90%, CXCL13 40%, BCL6 25%). As illustrated, PD1 expression shows a higher intensity in medium/large cells, and some PD1+ cells also tended to form "rosettes" around large lymphocytes (insert). Proliferative index using Ki67/Mib1 was evaluated a 15%, and no follicular dendritic cells network (pictures not shown). The diagnosis of SMLPD was confirmed thanks to molecular data; the presence of a monoclonal T-cell population. This case displayed an isolated TET2 pathogenic variant using TNGS. www.nature.com/scientificreports/ population in the lymph node. Cases no 7, 9 and 10, presented the same pathogenic variants in both samples, including TET2 and/or RHOA G17V hotspot.

Discussion
The differential diagnosis of cutaneous lymphomas with TFH expression and/or hyperplasia is frequently a diagnostic challenge in daily diagnosis work. The spectrum of cutaneous T-cell lymphomas arising from TFHcells extends from SMLPD to AITL 6 . Hyperplasia of TFH reactive T-cells is also frequently observe in cutaneous MZL 25 . The objective of the present study was to better characterize these different lesions (20 MZL, 13 SMLPD, 5 PCTFHL, and 11 cAITL) at the clinicopathological and molecular levels and to highlight tools to differentiate them.  www.nature.com/scientificreports/ The clinical presentation and histological appearance of the 49 cases described in this study were consistent with the literature 3,6,18,26 . In particular, MZL corresponded to erythematous papules/nodules (80%), or plaques (20%), which were most frequently located on the limbs (50%) and trunk (40%) 26 . Infiltrates in SMLPD could be separated into two patterns, as described by Beltzung et al.; (i) "Pattern 1" (n = 9/13, 69%): erythematous nodules, nodular/diffuse architecture, located on the head and neck (n = 4/9, 44%), followed by the trunk (n = 3/9, 33%) and upper extremities (n = 2/9, 23%); and (ii) "Pattern 2" (n = 4/13, 31%): erythematous-squamous plaques, lichenoid architecture, all located on the trunk 18 . Interestingly, loss of CD7 is described in the literature in 24% of SMLPD 18 . However, in this serie, none of the SMLPD cases showed CD7 loss, even partially. Although these data need to be confirmed in a larger cohort, it could be a tool to differentiate more aggressive forms of SMLPD, or even PCTFHL. Clinical presentation of PCTFHL was similar as described (multiple papules, plaques, and nodules of trunk/head) 5,6 , excepted that only one case presented a circulating monoclonal population (vs. 4/5 for Battistella et al.) 6 . Maculo-papular rash seems to be the classical presentation of cutaneous locations of AITL 3 .
In this study, the main objective was to compare these entities. That's why it was decided to include only cases with a monoclonal population, to try to include only "typical" cases and to limit the possibility of including inflammatory diseases, as, in the skin, dominant clones may also be found in a variety of benign dermatoses. Literature has already proved the interest of clonality assessment in the diagnosis of cutaneous lymphomas, the finding of a monoclonal population orienting the diagnosis in more than 80% of MZL 26 , more than 65% of SMLPD 18,27 , and all PCTFHL 6 . This study further emphasizes the importance of an integrated histomolecular diagnosis. Indeed, 4 MZL presented a PD1 expression with more than 30% of stained cells, making the differential diagnosis with SMLPD particularly difficult without molecular data. In these patients, a monoclonal B-cell population was found, without monoclonal T-cell population, and the diagnosis of MZL could be made. The study of clonality may therefore be helpful in these cases of MZL with TFH hyperplasia of the microenvironnement 10 . Moreover, this study also highlights the value of comparing molecular techniques in cases of suspected cutaneous localization of systemic lymphoma. The discovery of the same monoclonal population (AITL no 3) or same NGS pathogenic variants (AITL no 7, 9 and 10), in skin biopsy and lymph node can, indeed, allow to link two locations of the same disease.
Taken individually, TNGS also identified pathogenic variants helpful for the diagnosis in 42% of MZL and 64% of cAITL. Even if these cases do not represent a large majority, they are not negligible considering the importance of an optimal classification of these lesions for the management and prognosis.
Concerning AITL, most studies concern nodal locations, in which mutations of TET2 (52-76%), IDH2 (20-45%), DNMT3A (30-40%) and RHOA G17V (28-70%) seem frequent [11][12][13][14][15][16] . The only study currently published in cutaneous localizations is the one of Leclaire Alirkilicarslan et al., which included 41 patients and found IDH2 R172K/S and RHOA G17V mutations in 19% and 78% of cases respectively using PCR 17 . This study is the only one to study mutations using TNGS on a cohort of cutaneous localizations of AITL. Among them, 64% presented pathogenic variants involving TET2 (n = 7, 64%), RHOA (n = 4, 36%), and NOTCH1 (n = 1, 9%). Leclaire Alirkilicarslan et al. found 78% of RHOA G17V and 19% of cases with IDH2 R172 substitutions using www.nature.com/scientificreports/ PCR in a cohort of cAITL. In nodal AITL, mutations are much more frequent 11,12,15 ; TET2 SNV (76%), RHOA G17V (60-78%), and IDH2 R172 substitutions (19.5%). In the present study, the percentage of mutated cases was lower than described in the literature, and no IDH2 pathogenic variants were found. This may be explained by the small number of cases in the study or by the low density of tumoral cells in the samples. However, these molecular findings were confirmative of the diagnosis in 64% of cases, as in 4/5 cases with an atypical presentation (slight perivascular infiltrates, no identifiable atypical lymphocytes) in the study of Leclaire Alirkilicarslan et al. These data suggest that TNGS may represent a useful tool for the diagnosis of cAITL. The VAF of the SNV were lower in cAITL (average = 6.7%, median = 3.1%, ranging from 1.2 to 22%), compared to 14.2% in MZL and 45.2% in PCTFHL. It can be explained by the low cell density in these biopsies (less than 25% of the surface) or by clonal tumor heterogeneity, as already described in these entities 13,33 . Nevertheless, the sequencing quality remains optimal; the described variants are robust due to a minimal depth of 700× (Suppl. Table 3).
The second objective of this study was to decipher the spectrum of TFH lymphomas, including also lymphoproliferations and controversial intermediate forms. Concerning SMLPD, only one case (8%) presented a pathogenic variant of TET2, which may correspond to clonal hematopoiesis. Beltzung et al. reported a unique pathogenic variant of DNMT3A among 13 SMLPD, but this gene was not part of our panel 18 . Concerning PCT-FHL, two cases (40%) presented isolated pathogenic variants of ARID1A and SOCS1. These two mutated cases required aggressive treatment, whereas the two cases with an indolent disease did not present any detectable variant. ARID1A mutations are reported in numerous cancers and lymphomas and therefore does not seem really helpful in isolation 15,34 . SOCS1 was a class 3 of uncertain clinical significance 35,36 . This variant has already been described in TFH lymphomas 34 , but also in MF 37 . This finding was arguable, PCTFHL currently not being a recognized entity, and of difficult differential diagnosis with MF expressing TFH markers or Sezary syndrome 38,39 . In PCTFHL no 3 in particular, the clinical presentation as erythematosquamous plaques, the presence of epidermotropism, and the SOCS1 mutation may have suggested the diagnosis of MF or Sezary syndrome. Nevertheless, the absence of Sezary cells in the blood, the presence of interface dermatitis lesions, the polymorphism of the infiltrate (presence of 50% of B-cells and numerous plasma cells), and the intense expression of all TFH markers (PD1: 80%, CXCL13: 50%, BCL6: 20%, CD10: 10%) pleaded against this diagnosis. Within the panel studied, there does not appear to be a common or recurrent molecular profile between SMLPD, PCTFHL, or cAITL. The molecular abnormalities usually present in AITL were not found in SMLPD or PCTFHL. These data remain debatable and future studies will undoubtedly allow a better classification of these case. Indeed, one of the raised hypotheses was that the spectrum of TFH-lesions was potentially underpinned by a mutational spectrum. The frequency of mutations found in TNGS (8% in SMLPD, 40% in PCTFHL and 64% in cAITL) seems to go in the same direction as the prognosis of these lesions. As already described in nodal AITL, these data may suggest that RHOA mutation may be a secondary event in lymphomatous cells after other critical molecular events such as TET2 mutations that first occur in "premalignant" lymphocyte precursors 13,17,[40][41][42] . This hypothesis may agrees with the distribution of pathogenic variants in the present study and reinforces the theory of a spectrum of lymphoma derived from a TFH lymphocyte. These data will need to be confirmed in a larger cohort, gathering a larger number of PCTFHL cases. If accepted, the provisional PCTFHL entity will also need to be defined, in particular its exact terminology (lymphoproliferation or lymphoma), and diagnosis criteria. The controversial nature of these intermediate forms makes it difficult to include these patients in studies.
This study highlights some histological features to distinguish the main subtypes of cutaneous lymphomas expressing TFH markers, that might reasonably be considered in a differential diagnosis. It also underlines the interest of integrated histomolecular diagnosis, using clonality and NGS, to classify these pathologies and strength the hypothesis of a spectrum of cutaneous lymphomas arising from a T-follicular helper lymphocyte is, even if further studies on a more significant number of patients are required to draw firm conclusions. In particular, the provisional entity so-called PCTFHL will need to be defined thanks to larger cohorts, from clinical, histological, and molecular points of view, in order to determine if it related to lymphoproliferations (such as SMLPD), or to real lymphomas with TFH phenotype, by analogy to nodal lymphomas.

Data availability
All data generated or analyzed during this study are included in this published article (and its supplementary information files).